The central hypothesis of the project described in this Outstanding Investigator Award application is that regional tumor-targeted cytotoxic therapies, such as oncolytic poliovirus (PVS-RIPO) and the D2C7 PE38 immunotoxin (D2C7-IT), not only specifically target and destroy tumor cells, but in the process initiate immune events that promote an in situ vaccine effect, which can be amplified by immune checkpoint blockade to engender a long-term systemic immune response that effectively eliminates recurrent and disseminated glioblastoma multiforme cells (GBM). Ultimately, both agents may be used together providing two different antigenic targets and cytotoxicity mechanisms along with immune checkpoint blockade. In the initial years of the grant, transgenic mice carrying the human poliovirus gene will allow orthotopic growth of 3 astrocytic murine tumor cells that have been transfected with the human poliovirus receptor gene. The resultant tumors will be treated with PVS-RIPO and the nature and mechanism of the immune response will be characterized. Following that, the astrocytic animal tumors will be grown orthotopically, treated with PVS-RIPO, and then treated with anti-CTLA-4 and PD1 checkpoint inhibitors, both individually and together. A similar set of studies will be carried out with the D2C7 immunotoxin in 3 astrocytic murine tumor models that have been transfected with the murine version of the human epidermal growth factor receptor gene, which contains the murine epitope reactive with the D2C7-IT. A similar series of studies with orthotopic murine astrocytic tumors transfected with the D2C7 EGFR epitope will be treated with D2C7-IT, and individually and together, with the checkpoint inhibitors anti-CTLA-4 and PD1. The immune response will be characterized in depth, before and after treatment, with the checkpoint inhibitors. Following these animal studies, a similar series of human clinical trials in GBM patients will be carried out with treatment with either PVS-RIPO or D2C7-IT and the checkpoint inhibitors anti-CTLA-4 and PD1, both individually, and ultimately together. I believe the outcome of these studies will represent paradigm shifts in GBM treatment resulting in significant increases in high quality of life and overall survival.